Density separation of umbilical cord blood and recovery of hemopoietic progenitor cells: Implications for cord blood banking

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Abstract
Umbilical cord blood (CB) has been evaluated as a potential source of hematopoietic stem cells suitable for clinical use in the transplantation setting. Previous reports have documented a significant loss of progenitor cells by any manipulation other than cryopreservation. We have evaluated the feasibility of fractionating and cryopreserving CB samples with minimal loss of progenitor cells. We have compared various separation procedures based on different density gradients in the attempt to obtain the highest depletion of red blood cells (RBC) while maintaining the highest recovery of progenitor cells. We compared three different densities of Percoll (1.069 g/ml, 1.077 g/ml, 1.084 g/ml), sedimentation over poligeline (Emagel) and sedimentation over poligeline followed by separation over Ficoll/Hypaque (F/H). Separated samples (n = 25) were analyzed for recovery of CD34+ cells and progenitor cells (CFU-GEMM, BFU-E, CFU-GM). Separation by sedimentation over poligeline followed by F/H allowed the highest depletion of RBC (hematocrit of the final cellular suspension 0.4 ± 0.1%) while maintaining high recovery of CD34+ cells (85.3 ± 5.6%) and total recovery for CFU-GEMM, BFU-E and CFU-GM. After cryopreservation, recovery of clonogenic progenitors was 82% for CFU-GEMM, 94% for BFU-E, 82% for CFU-GM and 90% for colony-forming units (CFUs) after five weeks of long-term culture (LTC). We further evaluated the effect of stem cell factor (SCF) on the in vitro growth of hemopoietic progenitors and on replating efficiency. The presence of SCF significantly increased CFU-GEMM (14 ± 4 versus 49 ± 5, p < 0.0005) and CFU-GM (112 ± 18 versus 178 ± 19, p < 0.025), as well as the replating efficiency of CB progenitor cells (21 ± 3.5% versus 43.3 ±4.7%) and the number of CFC per replated colony (4.7 ± 3.5 versus 12.6 ± 5.3, p < 0.005). In conclusion, RBC depletion of umbilical CB can be accomplished with minimal loss of committed and primitive hemopoietic progenitors. This procedure may have important implications in the large-scale banking of CB as well as ex vivo expansion/gene therapy protocols.